1a. Optimizing immune reconstitution after HLA matched sibling SCT T cell depletion: In 2012 we initiated a new T cell depletion study to address some of the shortcomings of an earlier trial 07-H-0248. In this protocol 12-H-0028 the T cell depletion technique was changed so as to negatively select for CD34 cells using a CD3 CD19 antibody magnetic separation approach from Miltenyi Corporation under an investigator IND. The transplant product contains a mixed population of CD34 cells more mature progenitors and NK cells. It was anticipated that hematological reconstitution would be more rapid and robust with this method. Subjects received myeloablative conditioning with cyclophosphamide (60 mg/kg/dose x 2), fludarabine (25 mg/m2/dose x 5) and total body irradiation (12 Gy divided in 8 fractions, with lung shielding to 6 Gy). (ii) No planned donor lymphocyte infusion DLI given; ( DLI was reserved only for patients with graft failure or relapsed leukemia) (iii) Older patients receive 600cGy instead of 400cGy to improve engraftment since a number of older patients had engraftment failure in the previous protocol. This protocol accrued 20 patients and is now closed. This trial was terminated because of the high frequency of GVHD and graft failure which precluded its use as a platform for T cell therapy. In 2013 a new T cell depletion protocol 13-H-0144 initiated Peripheral Blood Stem Cell Allotransplantation for Hematological Malignancies Using Ex Vivo CD34 Selection - A Platform for Adoptive Cellular Therapy. This uses a modified version of protocol 07-H-0248 Currently 22 patient have received transplants. 5 developed grade II-IV acute GVHD, 1 died of transplant-related causes and four have died of relapsed leukemia. 1b. Ultra low dose IL-2 as a GVHD prophylaxis in haploidentical donor stem cell transplantation: We hypothesized that ULD IL-2 could serve as an immune-modulating agent for HSCT donors and recipients to prevent GVHD by expanding both Tregs and NK cells. We conducted a clinical trial in healthy volunteers to evaluate Tregs and NK cells following administration of three different doses of ULD IL-2 (protocol 11-H-0268). ULD IL-2 increased the Treg frequency and increased suppressive function. CD56bright NK cells with enhanced interferon-gamma (IFN-&#947;) production also expanded. Circulating IFN-&#947; induced protein 10 (IP-10) also increased. Gene expression analysis revealed significant highly-restricted gene changes involving FOXP3, IL-2RA, and CISH. This is the first study to describe global immune-modulation of ULD IL-2 in healthy subjects. Based on these results, we opened a clinical trial ULD-IL-2 therapy as GVHD prophylaxis in haplo-identical allogeneic stem cell transplantation (protocol 14-H-0180). The primary objective is safety and feasibility. We will also determine incidence of a- and cGVHD, engraftment, and standard transplant outcomes. The study adopts the HSCT method of Grosso et al where after conditioning, a DLI is followed by cyclophosphamide (to prevent GVHD) before CD34 infusion. Daily ULD-IL2 is given for 12wk starting day +1 after CD34+ cell infusion to expand Tregs and NK cells for GVHD prevention and GVL enhancement. Two patients have been transplanted. Both survive beyond 4 months and neither developed acute GVHD. 1c. Prevention of viral infection after SCT. We have developed a robust system to expand multivirus specific T cells recognizing CMV, EBV, Adenovirus and BK virus which commonly complicate the outcome after SCT. Using a peptide mix of common immunodominant antigens from these viruses we have induced multivirus specific T cells in high frequencies. The clinical protocol has been approved by the IRB and the clinical grade cell product multi-virus specific T cells (MVST) was approved by the FDA under an investigator IND. The objective of this Phase I/II protocol is first to establish safety of the MVST product and second to prevent CMV, EBV, BKV and adenovirus reactivation after SCT. Six patients undergoing T cell depleted SCT on protocol 13-H-0144 received an MVST infusion between day 7-30 post transplant. There was no infusional toxicity and preliminary observations suggest efficacy in reducing viral antigenemia and viral infection. 1d. Role of Th17 T cells in gut mucosal resistance to gram-negative bacteria. Emergence of gram-negative bacteria with resistance to multiple antibiotics is a significant challenge facing the healthcare system. Klebsiella pneumoniae is responsible for approximately 15% of nosocomial infections, predominantly affecting immunocompromised patients and in recent years multi-antibiotic resistant strains have emerged. SCT recipients are especially at risk from these resistant bacteria: In 2011-12 the NIH Clinical Center experienced an outbreak of multidrug resistant K. pneumoniae affecting 19 patients, most of whom had received stem cell transplants, 7 of whom died from the infection. While neutrophils play the predominant role in protection against Gram-negative bacteria emerging evidence suggests that T cells can orchestrate and regulate host defenses, especially at mucosal sites where Th17 cells physiologically reside. Th17 cells and their secreted cytokines bridge innate and adaptive responses and are involved in first line mucosal host. Recently, antigen-specific Th17 T cell recognizing K. pneumoniae have been described in an animal model of mucosal vaccination and infection. We have generated K. pneumoniae specific TH17 T cells and plan to develop a clinical-grade Th17 cells specific for Klebsiella pneumoniae from patients or HLA-matched normal donors for adoptive transfer as a secondary prevention of KPC-Klebsiella bacteremia in immunocompromised patients treated at the NIH Clinical Center. Completion of the proposed study will test a novel strategy of targeting antibiotic-resistant bacterial pathogens that cause post-transplant nosocomial infections. 2. Prevention and treatment of GVHD and post transplant complications with mesenchymal stromal cells: In 2013 we completed a phase I trial using third party, early passage, mesenchymal stromal cells (MSC) generated in the NIH Cell Processing Section for patients with steroid-refractory liver or gastrointestinal GVHD, tissue injury or marrow failure following SCT where we demonstrated both safety and efficacy of MSC infusions. These results confirmed that MSC are associated with rapid clinical responses of steroid refractory GVHD. A phase II trial evaluating the use of MSC infusions at the onset of systemic steroid treatment for acute GVHD has been initiated and the first patient receiving MSC showed rapid resolution of acute GVHD. The objective is to determine the effect of MSC on time to resolution of acute GVHD, progression to chronic GVHD and incidence of steroid refractoriness. All patients undergoing SCT at NIH are eligible for this protocol. 4. Long term follow-up: A prospective and comprehensive natural history study of allotransplant recipients surviving 3 to 22 years was established in 2005 (protocol 05-H-0130). We found, in over 150 patients, in the second decade after HSCT, there is some ability for repair of the T cell repertoire. By 10 years post HSCT pulmonary function (diffusion and alveolar ventilation) improves22 and bone mineral density increases (especially in younger individuals with a higher body mass index). Quality of life for survivors is excellent and correlates with less symptom distress. An emerging problem is premature cardiovascular ageing. Male survivors especially have a long-term persisting risk of cardiovascular events. Contrast-enhanced cardiac CT in 50 patients found coronary artery disease (CAD) in 45% of asymptomatic HSCT long-term survivors. These results show that cardiac CT imaging is a highly sensitive screening strategy for CAD in long-term survivors.